Atropisomer of pyrrole derivative

ABSTRACT

[Object] A prophylactic or therapeutic agent for a cardiovascular disease is provided. 
     [Means for Resolution] An atropisomer of a compound represented by the following general formula (I): 
                         
(wherein R 1  is a C1-C3 alkyl group or a hydroxy-C1-C3 alkyl group; and R 2  is a hydrogen atom or a C1-C3 alkoxy group).

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/JP2008/056907, filed Apr. 8, 2008, which claims priority fromJapanese Application No. 2007-101938, filed Apr. 9, 2007. Eachapplication is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to an atropisomer of a pyrrole derivativehaving excellent mineralocorticoid receptor antagonistic activity and toa prophylactic or therapeutic agent for hypertension, angina pectoris,acute coronary syndrome, congestive heart failure, nephropathy,arteriosclerosis, cerebral infarction, fibrosis or primary aldosteronismcontaining the same.

BACKGROUND ART

The mineralocorticoid receptor (MR) (aldosterone receptor) is known toplay an important role in regulating electrolyte balance and bloodpressure in the body (see, for example, Non-patent Document 1), andmineralocorticoid receptor antagonists such as spironolactone andeplerenone both of which have a steroidal structure are known to beuseful for the treatment of hypertension and heart failure.

Further, as a mineralocorticoid receptor antagonist having anonsteroidal backbone, a pyrrole derivative described in WO 2006/012642(Patent Document 1) is known. However, an atropisomer of a compoundhaving the general formula (I) of the invention is not known.

[Non-patent Document 1] Advances in Physiology Education, 26(1): 8-20(2002)

[Patent Document 1] WO 2006/012642

DISCLOSURE OF THE INVENTION Problems that the Invention is to Solve

The present inventors made efforts to develop an excellent prophylacticor therapeutic agent for cardiovascular disease and conducted intensivestudies of pharmacological activities of various pyrrole derivatives. Asa result, they found that atropisomers exist for a compound having thegeneral formula (I), and that one of the atropisomers showssignificantly excellent mineralocorticoid receptor antagonistic activity(in vitro and in vivo activities) and sustainability of agent effect,and further has excellent properties with respect to solubility, oralabsorption, blood concentration, metabolic stability, safety and thelike, and is useful as a pharmaceutical, preferably, as a prophylacticor therapeutic agent (particularly a therapeutic agent) for a diseasesuch as hypertension, angina pectoris, acute coronary syndrome,congestive heart failure, nephropathy, arteriosclerosis, cerebralinfarction, fibrosis, primary aldosteronism or heart disease, morepreferably, for congestive heart failure, nephropathy, hypertension orthe like, particularly preferably for hypertension, and thus, theinvention has been completed.

Means for Solving the Problems

The invention provides an atropisomer of a compound having the generalformula (I), having excellent mineralocorticoid receptor antagonisticactivity and a pharmaceutical containing the same [a prophylactic ortherapeutic agent (particularly a therapeutic agent) for hypertension,angina pectoris, acute coronary syndrome, congestive heart failure,nephropathy, arteriosclerosis, cerebral infarction, fibrosis, primaryaldosteronism or heart disease (more preferably, for congestive heartfailure, nephropathy and hypertension; particularly preferably forhypertension)].

That is, the invention relates to

(1) an atropisomer of a compound represented by the following generalformula (I):

(wherein R¹ is a C1-C3 alkyl group or a hydroxy-C1-C3 alkyl group; andR² is a hydrogen atom or a C1-C3 alkoxy group).

Further, the invention relates to the following aspects:

(2) an atropisomer which, between a pair atropisomers each of a compoundrepresented by the general formula (I), shows the more potentmineralocorticoid receptor antagonistic activity;

(3) the atropisomer according to (1) or (2), wherein R¹ is a methylgroup or a 2-hydroxyethyl group;

(4) the atropisomer according to any one of (1) to (3), wherein R² is ahydrogen atom or a methoxy group;

(5) the atropisomer according to (1) or (2), wherein R¹ is a2-hydroxyethyl group and R² is a hydrogen atom;

(6) the atropisomer according to (1) or (2), wherein R¹ is a methylgroup and R² is a hydrogen atom;

(7) the atropisomer according to (1) or (2), wherein R¹ is a2-hydroxyethyl group and R² is a methoxy group;

(8)(−)-1-(2-hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide;

(9)(+)-1,4-dimethyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide;

(10)(−)-1-(2-hydroxyethyl)-5-[4-methoxy-2-(trifluoromethyl)phenyl]-4-methyl-N-[4-(methylsulfonyl)phenyl]-1H-pyrrole-3-carboxamide;

(11) a pharmaceutical containing the atropisomer according to any one of(1) to (10) as an active ingredient;

(12) a prophylactic or therapeutic agent for a cardiovascular disease,containing the atropisomer according to any one of (1) to (10) as anactive ingredient;

(13) a prophylactic or therapeutic agent for hypertension, containingthe atropisomer according to any one of (1) to (10) as an activeingredient;

(14) a pharmaceutical composition comprising the atropisomer accordingto any one of (1) to (10) and a pharmacologically acceptable carrier.

Examples of the “C1-C3 alkyl group” in the above-mentioned generalformula (I) include linear or branched alkyl groups having 1 to 3 carbonatoms such as a methyl group, an ethyl group, an n-propyl group and anisopropyl group, and preferred is a methyl group.

The “hydroxy-C1-C3 alkyl group” in the above-mentioned general formula(I) means a group formed by substituting the above-mentioned “C1-C3alkyl group” with one hydroxy group. Examples thereof include a2-hydroxyethyl group, a 2-hydroxy-1-methylethyl group, a 2-hydroxypropylgroup and a 3-hydroxypropyl group, and preferred is a 2-hydroxyethylgroup.

The “C1-C3 alkoxy group” in the above-mentioned general formula (I)means a C1-C3 alkyloxy group formed from the above-mentioned “C1-C3alkyl group” and denotes a linear or branched alkoxy group having 1 to 3carbon atoms such as a methoxy group, an ethoxy group, an n-propoxygroup or an isopropoxy group, and preferred is a methoxy group.

The “atropisomer” refers to a structural isomer based on axial or planarchirality resulting from restricted rotation in the molecule. Thecompound having the general formula (I) of the invention has twoatropisomers derived from axial chirality which result from restrictedrotation about the bond between the phenyl group substituted at theortho position with a trifluoromethyl group and the substituted pyrrolering, due to steric hindrance. The “atropisomer” of the invention iseither one of the two atropisomers of the compound having the generalformula (I). However, it is preferably the atropisomer which shows themore excellent pharmacological activity, stability, in vivo kinetics,safety and the like and has favorable properties as a pharmaceutical.

Advantage of the Invention

The atropisomer of a compound having the general formula (I) of theinvention shows excellent mineralocorticoid receptor antagonisticactivity and high plasma concentration and blood retention, is excellentin pharmacological activity and in vivo kinetics such as oralabsorption, in vivo distribution and blood retention, and is also highlysafe to organs such as kidney and liver. Further, the atropisomer of acompound having the general formula (I) of the invention is very stable.For example, even after the atropisomer is treated in methanol at roomtemperature for 7 days or in an acetonitrile-phthalic acid buffer at 60°C. for 4 hours, racemization was not observed.

Therefore, the atropisomer of a compound having the general formula (I)of the invention is useful as, for example, a pharmaceutical, and isparticularly useful as a pharmaceutical for treating or preventingvarious cardiovascular diseases (preferably, hypertension, anginapectoris, acute coronary syndrome, congestive heart failure,nephropathy, arteriosclerosis, cerebral infarction, fibrosis, primaryaldosteronism or heart disease).

BEST MODE FOR CARRYING OUT THE INVENTION

The atropisomer of a compound having the general formula (I) of theinvention can be obtained by subjecting a racemic compound producedaccording to the method described in WO 2006/012642 to opticalresolution. The optical resolution of the atropisomers is essentiallythe same as that of enantiomers due to sp3 asymmetric carbons or thelike, and examples thereof include (1) a method by crystallization; (2)a method by enzymatic reaction; and (3) a method by chromatography.However, it is not limited to these. Hereinafter, representative opticalresolution methods will be described in detail.

-   (1) Optical Resolution by Crystallization-   (a) Preferential Crystallization Method

This is an optical resolution method using the property that a racemicmixture is spontaneously crystallized and can achieve optical resolutionwithout requiring an asymmetric element.

-   (b) Diastereomer Method

This is a method in which a chiral compound called an opticallyresolving agent is allowed to act on a racemic compound to derivatizethe compound into two diastereomers and these diastereomers areseparated by fractional crystallization utilizing the difference insolubility between these diastereomers. The optical purity can beincreased by repeating recrystallization. The objective enantiomer canbe obtained by removing the resolving agent from a thus obtained singlediastereomer. In the invention, a method in which fractionalcrystallization is performed after derivatization into covalentcrystalline diastereomers is preferred. For example, in the case where aracemic alcohol is to be resolved, the racemic alcohol is derivatizedinto diastereomer esters with chiral carboxylic acids, from which asparingly soluble diastereomer is taken out by recrystallization, andthe thus obtained single diastereomer ester is hydrolyzed, whereby anoptically active alcohol can be obtained.

-   (c) Inclusion Complex Method

This is an optical resolution method in which by using chiral hostmolecules and using one enantiomer of a racemic compound as a guestmolecule, an inclusion complex is diastereoselectively formed and theoptical purity is increased by recrystallization.

-   (d) Preferential Enrichment

This method is characterized in that enrichment of one enantiomer iscaused in a mother liquor by recrystallization of racemic crystals. Atthe same time, crystals having a low optical purity with chiralityopposite to that of the enantiomer in the mother liquor are deposited.

-   (2) Enzymatic Reaction

In the case where addition reaction to a racemic compound using anenzyme such as lipase is performed, the reaction of only one opticallyactive compound proceeds depending on the substrate. This is a methodwhich utilizes this property and in which, after the enzymatic reaction,the resulting product is separated and purified by recrystallization orchromatography, and thereafter the added functional group is removedunder an appropriate condition to obtain the objective optically activecompound. On the other hand, there is also a method in which a racemiccompound is specifically modified in advance, and the modified racemiccompound is subjected to enzymatic degradation reaction, and then, onlyone optically active compound is obtained in the same manner asdescribed above.

-   (3) Direct Optical Resolution by Chromatography

When a stationary phase incorporating an asymmetric element to which aderivative of a sugar or the like is attached is used as a support, adifference in retention time of chromatography is caused thereby toenable resolution. Utilizing this property, direct resolution can beperformed by high performance liquid chromatography with a chiralcolumn. As the chiral column, for example, CHIRALPAK AD-H, CHIRALCELOJ-RH (DAICEL) and the like can be exemplified.

In the case where the atropisomer of the invention is used as apharmaceutical, the atropisomer of a compound having the above-mentionedgeneral formula (I) can be administered as such or by mixing with anappropriate pharmacologically acceptable excipient, diluent or the likeorally in the form of a tablet, a capsule, a granule, a powder, a syrupor the like, or parenterally in the form of an injection, a suppository,a plaster, a preparation for external use or the like.

These preparations are produced by well-known methods using an additivesuch as an excipient, a lubricant, a binder, a disintegrant, anemulsifier, a stabilizer, a flavoring agent or a diluent.

The amount of atropisomer used varies depending on symptoms, age or thelike. However, it is preferably administered, in the case of oraladministration to a human adult at a dose of 0.02 mg/kg (preferably 0.1mg/kg) as a lower limit and 100 mg/kg (preferably 10 mg/kg) as an upperlimit, in the case of parenteral administration at a dose of 0.002 mg/kg(preferably 0.01 mg/kg) as a lower limit and 10 mg/kg (preferably 1mg/kg) as an upper limit one to six times a day depending on thesymptom.

Hereinafter, the invention will be described in more detail withreference to Examples, Test Examples and Preparation Examples. However,the Scope of the Invention is not limited to these.

EXAMPLES Example 1(+/−)-1,4-dimethyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide

The compound was synthesized by the method described in Example 16 of WO2006/012642.

¹H-NMR (400 MHz, CDCl₃) δ: 7.90 (2H, d, J=8.6 Hz), 7.83-7.80 (3H, m),7.70-7.58 (3H, m), 7.34 (1H, d, J=7.4 Hz), 7.30 (1H, s), 3.32 (3H, s),3.05 (3H, s), 2.09 (3H, s).

HR-MS (ESI) calcd for C₂₁H₂₀F₃N₂O₃S [M+H]⁺, required m/z: 437.1147,found: 437.1157.

Example 2 Optical Resolution of Compound of Example 1

Using 5 mL of an ethanol solution (4 to 6 mg/mL) of(+/−)-1,4-dimethyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide,resolution under the following HPLC conditions was performed 9 times,and 86 mg of Isomer A was obtained as a solid from a fraction containingIsomer A (t_(R)=11 min), and 87 mg of Isomer B was obtained as a solidfrom a fraction containing Isomer B (t_(R)=18 min).

For the separation by HPLC using a chiral column, the followingconditions were used.

Apparatus: Shimadzu Class-VP System (LC-8/SCL-10AVP/SPD-10AVP); column:CHIRALPAK AD-H (2 cm×25 cm) semi-fractionation column; flow rate: 8.0mL/min; elution solvent: ethanol (100%, isocratic); detection: UV (254nm)

Isomer A:(−)-1,4-dimethyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide

[α]_(D) ²¹: −18° (c=1.0, EtOH).

¹H-NMR (500 MHz, CDCl₃) δ: 7.92 (2H, d, J=8.3 Hz), 7.85-7.80 (3H, m),7.69-7.64 (2H, m), 7.61 (1H, t, J=7.3 Hz), 7.35 (1H, d, J=7.3 Hz), 7.31(1H, s) 3.33 (3H, s), 3.06 (3H, s), 2.10 (3H, s).

HR-MS (ESI) calcd for C₂₁H₂₀F₃N₂O₃S[M+H]⁺, required m/z: 437.1147,found: 437.1138.

Retention time: 4.1 min.

For an analysis by HPLC using a chiral column, the following conditionswere used. (Hereinafter, analysis was performed under the sameconditions. The retention time was determined by chiral HPLC.)

Analysis apparatus: Shimadzu Class-VP System (LC-10ADVP/SCL-10AVP/SPD-M10AVP/CT010ACVP/DGU12A); column: CHIRALPAK AD-H(0.46 cm×25 cm); flow rate: 1.0 mL/min; elution solvent: ethanol (100%,isocratic); detection: UV (254 nm)

Isomer B:(+)-1,4-dimethyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide

[α]_(D) ²²: +18° (c=1.2, EtOH).

¹H-NMR (500 MHz, CDCl₃) δ: 7.91 (2H, d, J=8.8 Hz), 7.85-7.80 (3H, m),7.68-7.64 (2H, m), 7.61 (1H, t, J=7.3 Hz), 7.35 (1H, d, J=7.3 Hz), 7.31(1H, s), 3.33 (3H, s), 3.06 (3H, s), 2.10 (3H, s).

HR-MS (ESI) calcd for C₂₁H₂₀F₃N₂O₃S [M+H]⁺, required m/z: 437.1147,found: 437.1153.

Retention time: 6.3 min.

Example 3(+/−)-1-(2-hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide

After methyl4-methyl-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxylate wasobtained by the method described in Example 16 of WO 2006/012642, thefollowing reaction was performed using this compound as a raw material.

Methyl 4-methyl-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxylate(1.4 g, 4.9 mmol) was dissolved in methanol (12 mL), and a 5 M aqueoussodium hydroxide solution (10 mL) was added thereto, and the resultingmixture was heated under reflux for 3 hours. After the mixture wascooled to room temperature, formic acid (5 mL) was added thereto to stopthe reaction. After the mixture was concentrated under reduced pressure,water (10 mL) was added thereto to suspend the resulting residue. Theprecipitated solid was collected by filtration and washed times withwater. The obtained solid was dried under reduced pressure, whereby4-methyl-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxylic acid (1.1g, 83%) was obtained as a solid. The thus obtained solid was suspendedin dichloromethane (10 mL), oxalyl chloride (0.86 mL, 10 mmol) was addedthereto, and the resulting mixture was stirred at room temperature for 2hours. After the mixture was concentrated under reduced pressure, theresidue was dissolved in tetrahydrofuran (10 mL), and4-(methylsulfonyl)aniline hydrochloride (1.0 g, 4.9 mmol) andN,N-diisopropylethylamine (2.8 mL, 16 mmol) were sequentially added tothe solution, and the resulting mixture was heated under reflux for 18hours. After the mixture was cooled to room temperature, the solvent wasdistilled off under reduced pressure, and acetonitrile (10 mL) and 3 Mhydrochloric acid (100 mL) were added to the residue. A precipitatedsolid was triturated, collected by filtration and washed with water, andthen, dried under reduced pressure, whereby4-methyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide(1.4 g, 89%) was obtained as a solid.

¹H-NMR (400 MHz, DMSO-d6) δ11.34 (1H, brs,), 9.89 (1H, s), 7.97 (2H, d,J=6.6 Hz), 7.87-7.81 (3H, m), 7.73 (1H, t, J=7.4 Hz), 7.65-7.61 (2H, m),7.44 (1H, d, J=7.8 Hz), 3.15 (3H, s), 2.01 (3H, s).

Sodium hydride (0.12 g, 3 mmol, 60% dispersion in mineral oil) wasdissolved in N,N-dimethylformamide (1.5 mL), and4-methyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide(0.47 g, 1.1 mmol) was added thereto, and then, the resulting mixturewas stirred at room temperature for 30 minutes. Then,1,3,2-dioxathiolane-2,2-dioxide (0.14 g, 1.2 mmol) was added thereto,and the resulting mixture was stirred at room temperature. After 1 hour,sodium hydride (40 mg, 1.0 mmol, oily, 60%) was added thereto again, andthe resulting mixture was stirred for 30 minutes. Then,1,3,2-dioxathiolane-2,2-dioxide (12 mg, 0.11 mmol) was added thereto,and the resulting mixture was stirred at room temperature for 1 hour.After the mixture was concentrated under reduced pressure, methanol (5mL) was added to the residue and insoluble substances were removed byfiltration, and the filtrate was concentrated again. To the residue,tetrahydrofuran (2 mL) and 6 M hydrochloric acid (2 mL) were added, andthe resulting mixture was stirred at 60° C. for 16 hours. The reactionwas cooled to room temperature, and then dissolved in ethyl acetate, andwashed with water and saturated saline. The organic layer was dried overanhydrous sodium sulfate and filtered. Then, the filtrate wasconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (ethyl acetate), whereby the objectivecompound (0.25 g, 48%) was obtained.

¹H-NMR (400 MHz, CDCl₃) δ: 7.89-7.79 (m, 6H), 7.66-7.58 (m, 2H), 7.49(s, 1H), 7.36 (d, 1H, J=7.4 Hz), 3.81-3.63 (m, 4H), 3.05 (s, 3H), 2.08(s, 3H).

HR-MS (ESI) calcd for C₂₂H₂₂F₃N₂O₄S [M+H]⁺, required m/z: 467.1252,found: 467.1246.

Anal. calcd for C₂₂H₂₁F₃N₂O₄S: C, 56.65; H, 4.54; N, 6.01; F, 12.22; S,6.87. found: C, 56.39; H, 4.58; N, 5.99; F, 12.72; S, 6.92.

Example 4 Optical Resolution of Compound of Example 3

Resolution was performed 4 times in the same manner as in Example 2,whereby 74 mg of Isomer C was obtained as a solid from a fractioncontaining Isomer C (t_(R)=10 min), and 71 mg of Isomer D was obtainedas a solid from a fraction containing Isomer D (t_(R)=11 min).

Isomer C:(+)-1-(2-hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide

[α]_(D) ²¹: +7.1° (c=1.0, EtOH).

¹H-NMR (400 MHz, CDCl₃) δ: 7.91 (s, 1H), 7.87-7.79 (m, 5H), 7.67-7.58(m, 2H), 7.51 (s, 1H), 7.35 (d, 1H, J=7.0 Hz), 3.78-3.65 (m, 4H), 3.05(s, 3H), 2.07 (s, 3H).

HR-MS (ESI) calcd for C₂₂H₂₂F₃N₂O₄S [M+H]⁺, required m/z: 467.1252,found: 467.1260.

Retention time: 4.0 min.

Isomer D:(−)-1-(2-hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide

[α]_(D) ²¹: −7.2° (c=1.1, EtOH).

¹H-NMR (400 MHz, CDCl₃) δ: 7.88-7.79 (m, 6H), 7.67-7.58 (m, 2H), 7.50(s, 1H), 7.36 (d, 1H, J=7.5 Hz), 3.79-3.65 (m, 4H), 3.05 (s, 3H), 2.08(s, 3H).

HR-MS (ESI) calcd for C₂₂H₂₂F₃N₂O₄S [M+H]⁺, required m/z: 467.1252,found: 467.1257.

Retention time: 4.5 min.

Example 5(+/−)-1-(2-hydroxyethyl)-5-[4-methoxy-2-(trifluoromethyl)phenyl]-4-methyl-N-[4-(methylsulfonyl)phenyl]-1H-pyrrole-3-carboxamide

The compound was synthesized by the method described in WO 2006/012642.

¹H-NMR (500 MHz, CDCl₃) δ: 7.91 (2H, d, J=8.3 Hz), 7.82 (2H, d, J=8.3Hz), 7.69 (1H, s), 7.46 (1H, s), 7.32 (1H, d, J=2.0 Hz), 7.28-7.27 (1H,m), 7.14 (1H, dd, J=8.3 and 2.0 Hz), 3.92 (3H, s), 3.82-3.66 (4H, m),3.06 (3H, s), 2.10 (3H, s).

HR-MS (ESI) calcd for C₂₃H₂₄F₃N₂O₅S [M+H]⁺, required m/z: 497.1358,found: 497.1361.

Example 6 Optical Resolution of Compound of Example 5

Resolution was performed 7 times in the same manner as in Example 2,whereby 50 mg of Isomer E was obtained as a solid from a fractioncontaining Isomer E (t_(R)=11 min), and 41 mg of Isomer F was obtainedas a solid from a fraction containing Isomer F (t_(R)=14 min).

Isomer E:(−)-1-(2-hydroxyethyl)-5-[4-methoxy-2-(trifluoromethyl)phenyl]-4-methyl-N-[4-(methylsulfonyl)phenyl]-1H-pyrrole-3-carboxamide

[α]_(D) ²²−1.3° (c=1.0, EtOH).

¹H-NMR (500 MHz, CDCl₃) δ: 7.90 (2H, d, J=8.3 Hz), 7.83-7.79 (3H, m),7.48 (1H, s), 7.32 (1H, d, J=2.4 Hz), 7.28-7.25 (1H, m), 7.14 (1H, dd,J=8.3 and 2.4 Hz), 3.92 (3H, s), 3.81-3.65 (4H, m), 3.06 (3H, s), 2.09(3H, s), 1.82 (1H, brs).

HR-MS (ESI) calcd for C₂₃H₂₄F₃N₂O₅S [M+H]⁺, required m/z: 497.1358,found: 497.1359.

Retention time: 4.1 min.

Isomer F:(+)-1-(2-hydroxyethyl)-5-[4-methoxy-2-(trifluoromethyl)phenyl]-4-methyl-N-[4-(methylsulfonyl)phenyl]-1H-pyrrole-3-carboxamide

[α]_(D) ²³: +1.6° (c=0.8, EtOH).

¹H-NMR (500 MHz, CDCl₃) δ: 7.91 (2H, d, J=8.8 Hz), 7.81 (2H, d, J=8.8Hz), 7.69 (1H, s), 7.46 (1H, s), 7.32 (1H, d, J=2.4 Hz), 7.28-7.25 (1H,m), 7.14 (1H, dd, J=8.3 and 2.4 Hz), 3.92 (3H, s), 3.82-3.66 (4H, m),3.05 (3H, s), 2.09 (3H, s).

HR-MS (ESI) calcd for C₂₃H₂₄F₃N₂O₅S [M+H]⁺, required m/z: 497.1358,found: 497.1340.

Retention time: 4.7 min.

Test Example 1

A plasmid pM-hMR-LBD which is obtained by ligation of a ligand-bindingdomain (LBD, corresponding to a region of about 308 amino acids at thecarboxy terminus) of human mineralocorticoid receptor (hMR,NM_(—)000901) to a DNA-binding domain (corresponding to a region of 147amino acids at the amino terminus) of a yeast transcription factor GAL4and expresses GAL4-hMR receptor was constructed. Using a reporterplasmid which has a sequence binding to the DNA-binding domain of GAL4(UAS sequence) and contains a luciferase gene (such as pFR-Luc, aplasmid available from Stratagene Cloning Systems), a reporter assay wasperformed.

The previously obtained plasmid pM-hMR-LBD and the reporter plasmid weretransfected into a human fetus-derived renal cell line HEK293 by thelipofection method. On the following day, the cells were treated withtrypsin and collected. A white 96-well plate (manufactured by Costar,Inc.) was prepared, and the cells were dispensed into each well in anamount of 95 μL using a DMEM medium containing 5% FBS (fetal bovineserum) which had been treated with activated charcoal.

As for each test compound, solutions obtained by dissolving the testcompound in dimethyl sulfoxide at predetermined concentrations wereused, and the solutions were appropriately diluted with medium and addedto the cells in the white 96-well plate to give a final concentration of0.1%. When the test compound was added, 1 nM aldosterone was allowed tobe present. A well group to which dimethyl sulfoxide was added wasassigned Control 1 group; and a well group to which 1 nM aldosterone wasadded was assigned Control 2 group. After the addition, the plate wasincubated overnight.

On the following day, the medium was removed, and a luciferase substrate(Wako Pure Chemical Industries, Ltd.) was prepared according to thepackage insert and added to each well in an amount of 50 μL, and then,the plate was stirred for about 30 minutes. The luminescence intensityof each well was measured using Analyst (manufactured by MolecularDevices Corporation) and taken as the luciferase activity. Theluciferase activity value of Control 1 group was taken as 0%, theluciferase activity value of Control 2 group was taken as 100%, andrelative luciferase activity values for the respective doses of the testcompound in the test compound addition group were plotted to create agraph. From the graph, the maximum value was calculated as Imax (%), andthe concentration of the test compound showing the value of Imax/2 wascalculated as ICmax50 (M). In Table 1, ICmax50 values are shown.

(Results) As shown in the following Table 1, the atropisomer of theinvention showed remarkable mineralocorticoid receptor antagonisticactivity as compared with the corresponding racemic compound.

TABLE 1 Test compound ICmax50 (nM) Imax (%) Compound of Example 1 13  95Isomer A >1000 N.D. ¹ Isomer B 2.6 123 Compound of Example 3 5.3 105Isomer C >1000 N.D. ¹ Isomer D 2.4  99 Compound of Example 5 5.3  97Isomer E 1.8 115 Isomer F >1000 N.D. ¹ ¹ Not Determined

Test Example 2

Cynomolgus monkeys (male) were used, and the monkeys were fasted fromone day before the test compound was administered. The administrationsample was prepared by adding a 0.5% MC (methyl cellulose) solution tothe test compound such that the dose was 3 mg/2 mL/kg. Eachadministration sample was administered to the stomach of the cynomolgusmonkey using a tube. After the sample was administered, about 5 mL ofwater was administered. Each administration sample was administered tothree cynomolgus monkeys in one group.

As for blood collection, about 1 mL of blood was collected from thefemoral vein before administration, and minutes and 1, 2, 4, 6, 8, 24and 48 hours after administration using an injection syringe treatedwith heparin. The collected blood was centrifuged (15,000×g, min, 4° C.)to obtain plasma. The obtained plasma was stored in a freezer (−20° C.)until pretreatment.

Preparation of Standard Solution and Internal Standard (IS) Solution:Each test compound was dissolved in acetonitrile, whereby a 1 mg/mLsolution of each test compound was prepared. A standard solution wasprepared by diluting each compound solution with acetonitrile. Further,sodium warfarin (Wako Pure Chemical Industries, Ltd.) was dissolved inacetonitrile to prepare a 500 ng/mL IS solution.

Pretreatment of Plasma Sample: 50 μL of the plasma sample was taken outand 50 μL of acetonitrile was added thereto. For a calibration curve, 50μL of each standard solution (acetonitrile solution) was added to 50 μLof the blank plasma. To all the samples, 150 μL of the IS acetonitrilesolution was added, and the mixture was stirred, and then centrifuged(about 1,800×g, 30 min, 4° C.). After filtration was performed withSirocco Protein Precipitation Plate (Waters Corporation), the filtratewas appropriately diluted with a mobile phase to prepare an LC-MS/MSanalysis sample.

Determination of Test Compound: The plasma concentration of each testcompound was analyzed by the LC-MS/MS method.

[HPLC Analysis Conditions]

HPLC: LC-10Avp series: Prominence (Shimadzu Corporation)

Column: X-Bridge RP18, 2.0 mm I.D.×50 mm, 2.5 μm (Waters Corporation)

Mobile phase: A=10 mM aqueous ammonium formate solution, B=acetonitrile

[MS/MS Analysis Conditions]

MS: API 4000 (AB/MDS SCIEX, Inc.)

Ionization Method Turbo ion spray (Positive or Negative)

Ionization Mode: Atmospheric pressure chemical ionization (APCI)

Detection Mode: MRM

Analysis: A pharmacokinetics parameter was calculated from the plasmaconcentration of each agent using WinNonlin Professional (Ver. 4.0.1,Pharsight Corporation). Incidentally, Noncompartment model was used as amodel for calculation of the parameter.

(Results) The compounds of Example 1, Isomer B of Example 2, Example 3,Isomer D of Example 4, Example 5 and Isomer E of Example 6 wereevaluated. As a result, as shown in Table 2, Isomer B, Isomer D andIsomer E, which are atropisomers with high activity demonstrated in TestExample 1 significantly improved the plasma concentration as comparedwith the compounds of Example 1, Example 3 and Example 5 which are thecorresponding racemic compounds, respectively.

TABLE 2 Test compound AUC ¹ (ng · h/mL) Cmax ² (ng/mL) Example 1 860 42Isomer B 3446 184 Example 3 7667 380 Isomer D 26390 1330 Example 5 33 2Isomer E 7187 681 ¹ AUC (ng · h/mL): Area under the plasma concentration(measured by LC-MS/MS method) versus time curve (0-48 hr.); ² Cmax(ng/mL): Maximum concentration

Formulation Example 1 Capsule

Isomer B 50.0 mg Lactose 128.7 Cornstarch 70.0 Magnesium stearate 1.3250 mg

Powder of the above formulation was mixed, and after the mixture waspassed through a sieve of 60 mesh, the powder was filled in a No. 3gelatin capsule of 250 mg to prepare a capsule.

Preparation Example 2 Tablet

Isomer D 50.0 mg Lactose 124.0 Cornstarch 25.0 Magnesium stearate 1.0200 mg

Powder of the above formulation was mixed and tabletted with atabletting machine to prepare a tablet (200 mg per tablet).

INDUSTRIAL APPLICABILITY

The atropisomer of a compound having the general formula (I) of theinvention shows particularly excellent pharmacological activities suchas a mineralocorticoid receptor antagonistic activity, anantihypertensive activity, a vasodilatory activity, a cardioprotectiveactivity, a nephropathy inhibitory activity, an antiarterioscleroticactivity and a diuretic activity, and also is highly safe, therefore itis useful as a prophylactic or therapeutic agent for hypertension,angina pectoris, acute coronary syndrome, congestive heart failure,nephropathy, arteriosclerosis, cerebral infarction, fibrosis or primaryaldosteronism.

The invention claimed is: 1.(−)-1-(2-hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide.2.(+)-1,4-dimethyl-N-[4-(methylsulfonyl)phenyl]-5-[2-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide.3.(−)-1-(2-hydroxyethyl)-5-[4-methoxy-2-(trifluoromethyl)phenyl]-4-methyl-N-[4-(methylsulfonyl)phenyl]-1H-pyrrole-3-carboxamide.4. A pharmaceutical consisting of the atropisomer according to any oneof claims 1, 2, or 3 as an active ingredient.
 5. A pharmaceuticalcomposition comprising the atropisomer according to any one of claims 1,2, or 3 and a pharmacologically acceptable carrier.
 6. The compositionof claim 5 in the form of a tablet, a capsule, a granule, a powder, asyrup, an injection, a suppository, or a plaster.